1. Field of the Invention
This invention relates to a system and method for producing proteins, and more particularly this invention relates to a cell-free system and cell-free method for producing and maintaining heterologous membrane proteins in their native forms.
2. Background of the Invention
The cell membrane serves as the interface between an organism and its environment, and internal membranes in eukaryotic organisms separate functional compartments within cells. Proteins inserted in these membranes carry out the essential functions of the cell: biological processes such as uptake of nutrients, excretion of wastes, generation of energy, and signal transduction.
The functions performed by membrane proteins are extremely important for all organisms. Despite the fact that membrane proteins represent approximately 30% of every genome and comprise more than 60% of all drug targets, only about 100 unique membrane protein structures have been determined to date, in contrast with unique structures representing approximately 10,000 soluble protein families.
A major factor influencing the paucity of membrane protein structures is that the expression levels of membrane proteins in native tissue are generally low. While many membrane proteins have been isolated in functional form from their native host organisms, purification in such cases is highly protein-specific, is not adaptable to high-throughput methodologies, and rarely yields the amounts of pure membrane proteins that are needed for extensive biochemical studies and crystallization trials.
Since the natural abundance of many membrane proteins is low and the purification process is daunting, recombinant systems are often employed now to overexpress membrane proteins. Escherichia (E.) coli-based systems are used most commonly for the heterologous expression of soluble proteins, as they offer many advantages such as simplicity, low cost and rapid growth. They suffer limitations, however, especially when applied to the expression of non-E. coli membrane proteins. Significantly, native E. coli strains do not have adequate space in their membranes to accommodate heterologously-expressed membrane proteins, as noted in Arechaga, I., Miroux, B., Karrasch, S., Huijbregts, R., de Kruijff, B., Runswick, M. J., and Walker, J. E. (2000) “Characterization of new intracellular membranes in Escherichia coli accompanying large scale over-production of the b subunit of F(1)F(o) ATP synthase.” FEBS Letters 482, 215-219, and Miroux, B., and Walker, J. E. (1996) “Over-production of proteins in Escherichia coli: Mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels.” Journal of Molecular Biology 260, 289-298.
Because the host cell's own membrane proteins already occupy a defined proportion of the cell's membranes, the availability of any remaining membrane space severely limits the absolute number of copies of the target protein which can be properly inserted into the cell membranes.
In addition, a high level of expression in E. coli can saturate the secretory machinery for integration of the heterologous protein into the membrane, often resulting in cell death, degradation of the target protein, or precipitation of the heterologously-expressed membrane protein as inclusion bodies (insoluble aggregates within cells). (Kiefer et al., Biochemical Society Transactions 27, 908-912 (1999); Korepanova et al., Protein Science 14:148-158 (2005); and Columbus et al., Protein Science 15:961-975 (2006).
Overall success of an in vivo Rhodobacter membrane protein expression system is encouraging, per U.S. Pat. No. 6,465,216 awarded to the inventors on Oct. 15, 2002, and incorporated herein by reference. However, the inventors have observed that expression of some target proteins has a negative impact on cell growth rate. Also, some target membrane proteins are expressed early in the auto-induction process but then disappear as the cell density increases, suggesting proteolysis.
Many eukaryotic protein expression systems are also available and have been employed for the production of membrane proteins. However, they suffer from many of the same limitations and are cumbersome and expensive for the preparation of the quantities of membrane proteins that are necessary for structure determination experiments.
Rhodobacter (R.) cell free extracts also have been used to produce native membrane proteins, as reported in Troschel D, Eckhardt S, Hoffschulte H K and Muller M (1992) Cell-free synthesis and membrane integration of the reaction center subunit H from Rhodobacter capsulatus. FEMS Microbiol Lett 91:129-133. However, these proteins were localized to Rhodobacter ICM vesicles if (and only if) the vesicles were added cotranslationally; that is, the ICM must be present during protein synthesis for efficient membrane incorporation. More importantly, Rhodobacter extracts have not been used to produce heterologous proteins.
The need exists in the art for a cell-free system and method which enables production of significant quantities of heterologous membrane proteins in functional form. The system and method should be compatible with all subsequent steps of sequestration, solubilization, stabilization and purification of the target membrane protein.